PROJECT SUMMARY/ABSTRACT The endogenous antioxidant glutathione (GSH) confers cells with the ability to resist stress, maintain survival, and properly regulate fundamental signaling pathways. The levels of GSH within a tissue, as well as the proportion of its reduced and oxidized forms, appear to be innate, demonstrating that eukaryotes inherit the relative capacity to synthesize and metabolize GSH. Despite this apparent genetic effect, the genetic regulation of the GSH system remains poorly defined. Instead, knowledge is currently limited to a small number of canonical GSH genes such as glutathione reductase (Gr) and glutathione peroxidase-1 (Gpx-1). Our preliminary studies revealed that the genetic regulation of this system is actually more complex and may involve a novel set of genes. Those preliminary efforts were based on in silico methods that are at times limited in power, so it is now paramount to perform high precision gene mapping to validate our newly discovered loci, and to identify previously overlooked loci. In the current project, we will accomplish those crucial tasks by testing our central hypothesis: that the GSH system is regulated by genetic variation within i) canonical GSH genes, including Gr and Gpx-1, and ii) novel genes, such as the RAR-related orphan receptor ? (Ror?), whose functions are external to the basic GSH system, and whose number we expect to exceed that of canonical GSH genes. We will test the hypothesis with a strategy that couples a forward genetics approach with the innovative Diversity Outbred (DO) mouse stock, which models the genetic diversity found in humans, and a reverse genetics approach based on novel mouse models created with CRISPR/Cas9 technology. We will address the following specific aims: 1) to quantify the heritability of core GSH phenotypes in a genetically diverse population; 2) to define genomic regions associated with the GSH system, and delineate shared and tissue-specific loci; and 3) to prioritize candidate genes, and initiate functional analyses of the most compelling candidates. These studies will define the fundamental genetic architecture of an indispensable biochemical system that governs cellular stress resistance and survival. Knowledge gained from these efforts will inform a series of future clinical and mechanistic studies aimed at understanding the impact of GSH genes on cellular damage during stress, and the data will build a foundation for innovative therapies to maintain tissue integrity in patients with degenerative diseases, thereby increasing their health spans and improving their qualities of life.